Changes in pH have a marked influence on the CA++ sensitivity of the contractile apparatus, such that a higher (CA++) is required for activation as pH is decreased. In explanation, however, studies on troponin (Tn) fail to indicate any competition between H+ and CA++ for the binding sites thought to be involved in control of contraction. These effects of pH can be explained by an effect of H+ on the net charge, and hence the electrostatic potential, at the surface of the thin filament. This potential will, in turn, influence the concentration of CA++ in the region of the Tn binding sites. This simple model can quantitatively predict the observed shift of the relative force-pCa relation as pH is varied in the range 5.5 - 7.5, if the net filament charge in this pH range is dominated by groups with a pK similar to histidine imidazoles. The aim of this proposal is to test this model further by observing the influence of changes in pH, Mg++, and ionic strength on the activation of skinned muscle fibers by CA++, monitoring both mechanical (force vs pCa) and biochemical (ATPase vs pCa) parameters. In addition, the influence of specific chemical modification of imidazole side chains with diethyl-pyrocarbonate on the force- the ATPase-pCa relations will be observed. Single skinned fibers from both frog and rabbit muscle will be used. Force will be measured with a conventional strain gauge; ATPase rates will be measured either by high pressure liquid chromatography, monitoring the appearance of creatine from the ATP-phosphocreatine reaction, or by a fluorometric technique, observing the oxidation of NADH in a conventional linked enzyme system involving phosphoenolpyruvate as the high-potential phosphoryl group donor. Regardless of the validity of the model, the proposed experiments will forward the longer-term objectives of elucidating the molecular bases of the activation process. Furtermore, the relation between force and ATPase under this variety of conditions will provide tests for any cross-bridge model which attempts to link the mechanical and biochemical events of the cross-bridge cycle. Lastly, these experiments are of clinical interest since the negative inotropy observed with ischemica, most especially in cardiac muscle, has been attributed to alteration of the CA++ -activation process resulting from intracellular acidosis.